plasmid pnfat luc  (Addgene inc)

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Epitope-directed selection of GPCR nanobody ligands with evolvable function.

doi: 10.1073/pnas.2423931122

Figure Lengend Snippet: Fig. 4. Engineering of AT206. (A). Overlay of AT206, AT209, and AT118-bound AT1R. All three CDRs are oriented similarly. Image is colored as follows: CDR1 (AT206, orange; AT209, purple; AT118, dark blue), CDR2 (light green), CDR3 (magenta), AT1R (light blue), ECL1 (yellow), ECL2 (light pink), and ECL3 (red). Unlike the allosteric parent nanobody AT206, the AT118-AT206 chimera suppresses AngII-mediated (B) Gαq signaling, as measured through the NFAT transcriptional reporter and (C) β-arrestin 2 recruitment, as measured through the TANGO assay. Signaling assay data are presented as mean ± SEM from three experiments.

Article Snippet: The FLAG- AT1R HEK293T line was grown in DMEM + 10% FBS and transfected with 3.5 μg of a NFAT luciferase reporter plasmid [gift from Toren Finkel (National Institutes of Health, Bethesda, MD), Addgene plasmid #10959 (45)] with FuGENE (3 μL/μg DNA) in a 10 cm plate.

Techniques: